Development of an accurate lateral flow immunoassay for PEDV detection in swine fecal samples with a filter pad design.

Porcine epidemic diarrhea virus (PEDV), as the main causative pathogen of viral diarrhea in pigs, has been reported to result in high morbidity and mortality in neonatal piglets and cause significant economic losses to the swine industry. Rapid diagnosis methods are essential for preventing outbreaks and transmission of this disease. In this study, a paper-based lateral flow immunoassay for the rapid diagnosis of PEDV in swine fecal samples was developed using stable color-rich latex beads as the label. Under optimal conditions, the newly developed latex bead-based lateral flow immunoassay (LBs-LFIA) attained a limit of detection (LOD) as low as 103.60 TCID50/mL and no cross-reactivity with other related swine viruses. To solve swine feces impurity interference, by adding a filtration unit design of LFIA without an additional pretreatment procedure, the LBs-LFIA gave good agreement (92.59%) with RT-PCR results in the analysis of clinical swine fecal samples (n = 108), which was more accurate than previously reported colloidal gold LFIA (74.07%) and fluorescent LFIA (86.67%). Moreover, LBs-LFIA showed sufficient accuracy (coefficient of variance [CV] < 15%) and stable (room temperature storage life > 56 days) performance for PEDV detection, which is promising for on-site analysis and user-driven testing in pig production system. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s44149-021-00029-1.

CD8 cell counting in whole blood by a paper-based time-resolved fluorescence lateral flow immunoassay.

The number of CD8+ T lymphocytes (CD8 cells) in peripheral blood can directly reflect the immune status of the body and is widely used for auxiliary diagnosis and prognostic evaluation of diseases. There is an urgent need to develop a simple CD8 cell-counting platform to meet clinical needs. Our group designed a paper-based cell-counting method based on a blocking competition strategy. In addition, we developed a time-resolved fluorescence-blocking competitive lateral flow immunoassay (TRF-BCLFIA) for point-of-care CD8 cell counting that functions by measuring europium nanoparticle (EuNP)-labeled CD8 antibody probes that are not captured by CD8 cells, and we indirectly calculated the concentration of CD8 cells in samples. Within 30 min, four operation steps can provide an accurate CD8 cell count for a 75-µL whole-blood sample, and this approach can be implemented on a handheld device. The TRF-BCLFIA reliably quantified CD8 cells in whole-blood samples, in which the assay exhibited a linear correlation (R2 = 0.989) readout for CD8 cell concentrations ranging from 137 to 821 cells/µL. To validate this approach, our newly developed CD8 cell-counting tool was used to assess 33 tumor patient blood samples. The results showed a high consistency with a flow cytometry-based absolute count. This analysis approach is a promising alternative for the costly standard flow cytometry-based tools for CD8 cell counting in tumor patients in community clinics, small hospitals, and low medical resource regions. This technology would deliver simple diagnostics to patients anywhere in the world, regardless of geography or socioeconomic status.

On-site differential diagnostic detection of HP-PRRSV and C-PRRSV using EuNPs-mAb fluorescent probe-based immunoassay.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused worldwide economic losses in the swine industry. Pigs infected with highly pathogenic (HP)-PRRSV display more severe symptoms than those infected with classical (C)-PRRSV. A rapid, sensitive, and reliable detection method to distinguish between HP-PRRSV and C-PRRSV is needed. In this study, we prepared a monoclonal antibody from a hybridoma that can distinguish HP-PRRSV(including TP, QJ, LQ, JN-HS, and TY strain) from C-PRRSV (CH-1A strain) using cell surface-fluorescence immunosorbent assays (CSFIA). Based on this monoclonal antibody (4D5), we developed a europium microsphere-based lateral flow immunochromatographic strip (EuNPs-LFICS) for the differential diagnostic detection of HP-PRRSV and C-PRRSV. Under optimized conditions, the method was rapid (15 min), sensitive (LOD: 2.57 ng mL-1, 606 TCID50/0.1 mL), selective for HP-PRRSV detection, and quantitative (DLR: 3.56-228 ng mL-1). In clinical samples, the EuNPs-LFICS assay was largely consistent with PCR results, indicating its practical clinical application.

A new method for rapid screening of hybridoma cell clones secreting paired antibodies using sandwich cell surface fluorescence immunosorbent assay.

Traditional methods of screening antibody pairs through ELISA-based methods are time-consuming and burdensome, which is not conducive for the rapid establishment of antigen detection methods. Hence, we developed a new method based on the sandwich cell surface fluorescence immunosorbent assay (SCSFIA) for rapid screening of paired antibodies. In this method, the capture antibodies were anchored to the hybridoma cells membrane through the lipid derivative Oleyl-PEG4000-NHS. Goat anti-mouse antibodies (blocking agent) were added to block the Fc fragment of the capture antibodies. The capture antibodies' Fab fragment can specifically bind the added antigen and form the capture antibodies-antigens complex (immunocomplexes). If the antibodies secreted by hybridoma cells could recognize the immunocomplexes. A double antibody sandwich structure would form on the cell surface based on the specific binding of antigens and antibodies. The hybridoma cells would be stained with anti-mouse IgG-Fc-FITC antibodies. We first used anti-pseudorabies virus (anti-PRV) cells and anti-porcine epidemic diarrhea virus (anti-PEDV) cells to verify the new method. Then, we used this method to successfully screen 5 hybridoma cell clones secreting paired antibodies against Avian influenza A (H7N9) virus within 15 days after fusion. These results showed that this method is suitable for the screening of paired antibodies in a variety of virus. Compared with the traditional method of obtaining paired antibodies, this method can greatly shortens the time needed to screen paired antibodies and improves screening efficiency, indicating that it is a promising method for paired antibodies discovery.

Identification of conserved tyrosine residues important for gibberellin sensitivity of Arabidopsis RGL2 protein.

DELLA proteins are regulators in the signaling pathway of gibberellin (GA), a plant growth regulator of diverse functions. GA typically induces the degradation of DELLA proteins to overcome their repressive roles in growth and development. We have previously evaluated the likely roles of Ser-Thr phosphorylation of DELLA proteins in GA signaling (Hussain et al., Plant J 44:88-99, 2005). Here we report that four DELLA proteins of Arabidopsis, namely GAI, RGL1, RGL2 and RGL3, expressed in tobacco BY2 cells, are degradable by GA. Both, proteasome inhibitor and protein tyrosine (Tyr) kinase inhibitors, strongly inhibit GA-induced DELLA degradation whereas phospho-Tyr phosphatase inhibitors have no effect, suggesting that Tyr phosphorylation is critical in GA-induced DELLA degradation. Mutation of eight conserved Tyr residues of RGL2 into alanine shows four mutant proteins (Y52A, Y89A, Y223A and Y435A) are resistant to GA-induced degradation. Substitution of these four critical Tyr residues into negatively charged glutamate (Y --> E) also resulted in stabilization of these mutants against GA treatment. However, further mutation of these four Tyrs into conservative phenylalanine (Y --> F) rendered the mutant proteins sensitive to GA like the wild-type RGL2. Since Y --> E mutations sometimes mimic phosphor-Tyr whereas Y --> F mutations render the protein unphosphorylatable at these Tyr sites, we conclude that these four conserved Tyrs, despite being critical for GA-sensitivity, are unlikely to be sites of Tyr phosphorylation but instead play important roles in maintaining the structure integrity of RGL2 for GA-sensitivity.

Gibberellin mobilizes distinct DELLA-dependent transcriptomes to regulate seed germination and floral development in Arabidopsis.

Severe Arabidopsis (Arabidopsis thaliana) gibberellin (GA)-deficient mutant ga1-3 fails to germinate and is impaired in floral organ development. In contrast, the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutant confers GA-independent seed germination and floral development. This fact suggests that GA-regulated transcriptomes for seed germination and floral development are DELLA dependent. However, it is currently not known if all GA-regulated genes are GA regulated in a DELLA-dependent fashion and if a similar set of DELLA-regulated genes is mobilized to repress both seed germination and floral development. Here, we compared the global gene expression patterns in the imbibed seeds and unopened flower buds of the ga1-3 mutant with that of the wild type and of the ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutant. We found that about one-half of total GA-regulated genes are apparently regulated in a DELLA-dependent fashion, suggesting that there might be a DELLA-independent or -partially-dependent component of GA-dependent gene regulation. A cross-comparison based on gene identity revealed that the GA-regulated DELLA-dependent transcriptomes in the imbibed seeds and flower buds are distinct from each other. Detailed ontology analysis showed that, on one hand, DELLAs differentially regulate the expression of different individual members of a gene family to run similar biochemical pathways in seeds and flower. Meanwhile, DELLAs control many functionally different genes to run specific pathways in seeds or flower buds to mark the two different developmental processes. Our data shown here not only confirm many previous reports but also single out some novel aspects of DELLA functions that are instructive to our future research.

Identification of the conserved serine/threonine residues important for gibberellin-sensitivity of Arabidopsis RGL2 protein.

The DELLA proteins GAI, RGA, RGL1 and RGL2 in Arabidopsis are plant growth repressors, repressing diverse developmental processes. Studies have shown that gibberellin (GA) attenuates the repressive function of DELLA proteins by triggering their degradation via the proteasome pathway. However, it is not known if GA-induced protein degradation is the only pathway for regulating the bioactivity of DELLA proteins. We show here that tobacco BY2 cells represent a suitable system for studying GA signaling. RGL2 exists in a phosphorylated form in BY2 cells. RGL2 undergoes GA-induced degradation, and this process is blocked by proteasome inhibitors and serine/threonine phosphatase inhibitors; however, serine/threonine kinase inhibitors had no detectable effect, suggesting that dephosphorylation of serine/threonine is probably a prerequisite for degradation of RGL2 via the proteasome pathway. Site-directed substitution of all 17 conserved serine and threonine residues showed that six mutants (RGL2(S441D, RGL2(S542D), RGL2(T271E), RGL2(T319E), RGL2(T411E) and RGL2(T535E)) mimicking the status of constitutive phosphorylation are resistant to GA-induced degradation. This suggests that these sites are potential phosphorylation sites. A functional assay based on the expression of GA 20-oxidase revealed that RGL2(T271E) is probably a null mutant, RGL2(S441D), RGL2(S542D), RGL2(T319E) and RGL2(T411E) only retained about 4-17% of the activity of the wild type RGL2, whereas RGL2(T535E) retained about 66% of the activity of the wild type RGL2. However, expression of GA 20-oxidase in BY2 cells expressing these mutant proteins is still responsive to GA, suggesting that the stabilization of RGL2 protein is not the only pathway for regulating its bioactivity.

Loss of function of four DELLA genes leads to light- and gibberellin-independent seed germination in Arabidopsis.

The Arabidopsis severe gibberellin-deficient mutant ga1-3 does not germinate even when the optimal light and temperature conditions are provided. This fact suggests that (1) gibberellin (GA) is absolutely necessary for the germination of an intact seed and (2) the ga1-3 mutant can be used as a good system to identify factors that repress seed germination. In this report, using ga1-3 mutation as the genetic background, we confirm that RGL2, one member of the DELLA family, encodes the predominant repressor of seed germination in Arabidopsis and show that the other DELLA genes GAI,RGA and RGL1 enhance the function of RGL2. More importantly, we show that ga1-3 seeds lacking RGA, RGL1 and RGL2 or GAI, RGL1 and RGL2, confer GA-independent germination in the light but not in the darkness whilst ga1-3 seeds lacking GAI, RGA and RGL2 germinate both in the light and darkness. This suggests that the destabilization or inactivation of RGA and GAI is not only triggered by GA but also possibly by light. In addition, ga1-3 seeds lacking in all the aforementioned four DELLA genes have elongated epidermal cells and confer light-, cold- and GA-independent seed germination. Therefore, DELLA proteins likely act as integrators of environmental and endogenous cues to regulate seed germination.

Gibberellin regulates Arabidopsis floral development via suppression of DELLA protein function.

The phytohormone gibberellin (GA) regulates the development and fertility of Arabidopsis flowers. The mature flowers of GA-deficient mutant plants typically exhibit reduced elongation growth of petals and stamens. In addition, GA-deficiency blocks anther development, resulting in male sterility. Previous analyses have shown that GA promotes the elongation of plant organs by opposing the function of the DELLA proteins, a family of nuclear growth repressors. However, it was not clear that the DELLA proteins are involved in the GA-regulation of stamen and anther development. We show that GA regulates cell elongation rather than cell division during Arabidopsis stamen filament elongation. In addition, GA regulates the cellular developmental pathway of anthers leading from microspore to mature pollen grain. Genetic analysis shows that the Arabidopsis DELLA proteins RGA and RGL2 jointly repress petal, stamen and anther development in GA-deficient plants, and that this function is enhanced by RGL1 activity. GA thus promotes Arabidopsis petal, stamen and anther development by opposing the function of the DELLA proteins RGA, RGL1 and RGL2.